Molecular Pathogenesis of Vibrio vulnificus

超出所提供的空间。创伤弧菌是一种革兰氏阴性菌，可在易感人群中引起暴发性疾病：食用牡蛎后的败血症和海水伤口感染。主要的诱发因素是铁超载。败血症和伤口感染的死亡率分别为77%和15%，患者可在接触细菌24小时内死亡。创伤弧菌具有很强的侵袭性，在宿主组织中复制迅速，导致大量细菌和广泛的组织损伤。我们使用皮下接种，铁葡聚糖处理的小鼠显示了广泛的组织损伤，类似于人类疾病，并区分了强毒的临床菌株和弱毒的牡蛎菌株。毒力极强的创伤弧菌在小鼠体内复制极快，并对PMNs具有抗性。我们的假设是创伤弧菌在宿主组织中的快速生长和对宿主吞噬防御的抵抗力使其数量达到很高，并通过多因子毒素造成组织损伤。我们的初步结果使我们能够使用遗传工具对动物模型中的每个因素进行解剖。我们建议继续使用分子遗传学方法来鉴定V. vu/nificus的毒力因子。具体目标是：1)利用特征标记诱变（STM）、PhoA融合/插入诱变和体内选择互补自然发生的减毒突变相结合的方法鉴定创伤弧菌的毒力基因；2)利用标记质粒与右旋铁处理的小鼠模型区分毒力基因对生长、杀伤和损伤的影响，并利用体外模型更详细地表征毒力表型。3)完成关于重要毒力基因的科赫假设的分子版本。在目标1中，我们将主要使用STM来获得与pmn快速生长或逃避有关的基因突变。我们将使用PhoA融合/插入诱变来鉴定编码分泌蛋白的基因。在目的2中，在区分突变对宿主生长和杀伤的影响之后，我们将通过检查组织病理学来表征损伤，并通过感染中性粒细胞减少的小鼠来检查弧菌与pmn的相互作用。体外试验将包括分析营养缺陷、铁获取、补体耐药、PMN耐药和细胞毒性。这些研究将阐明创伤弧菌引起的暴发性侵袭性疾病与快速复制、逃避防御和损害宿主组织有关的机制。网站性能 ======================================== 节结束 ===========================================

EXCEED THE SPACE PROVIDED. Vibrio vulnificus is a gram-negative bacterium that causes fulminating diseases in susceptible humans: septicemia after ingestion of oysters and wound infection from sea water. The major predisposing factor is iron overload. The mortality rates for septicemia and wound infection are 77% and 15%, respectively, and patients can die within 24 hours of contact with the bacteria. V. vulnificus is highly invasive and replicates rapidly in host tissues, leading to high numbers of bacteria and extensive tissue damage. Our use of subcutaneously inoculated, iron dextran-treated mice revealed extensive tissue damage that resembles human disease and differentiated virulent clinical strains from less virulent oyster strains. Virulent V. vulnificus replicated extremely rapidly in the mice and were resistant to PMNs. Our HYPOTHESIS is that the rapid growth rate of V. vulnificus in host tissues and resistance to host phagocytic defenses enable the bacteria to reach high numbers and cause tissue damage with multifactorial toxins. Our preliminary results have enabled the dissection of each of these factors in the animal model using genetic tools in use by us. We propose to continue to use a molecular genetic approach to identify virulence factors of V. vu/nificus. The SPECIFIC AIMS are to: 1) Use a combination of signature-tagged mutagenesis (STM), PhoA fusion/insertion mutagenesis, and in vivo selection for complementation of naturally occurring attenuating mutations to identify virulence genes of V. vulnificus, 2) Use a marker plasmid with the iron dextran-treated mouse model to differentiate the effects of virulence genes on growth, killing, and damage, and use in vitro models to characterize the virulence phenotypes in more detail, and 3) Complete the molecular version of Koch's postulates for important virulence genes. In aim 1 we will primarily use STM to obtain mutations in genes involved with rapid growth or evasion of PMNs. We will use PhoA fusion/insertion mutagenesis to identify genes encoding secreted proteins. In aim 2 after differentiating effects of mutations on growth in versus killing by the host, we will characterize damage by examining histopathology and examine the interaction of vibrios with PMNs by infecting neutropenic mice. In vitro assays will involve analysis of auxotrophy, iron acquisition, complement resistance, PMN resistance, and cytotoxicity to cell culture. These studies will elucidate mechanisms of fulminating, invasive disease caused by V. vulnificus as related to rapid replication, evasion of defenses, and damage to host tissues. PERFORMANCE SITE ========================================Section End===========================================